In plants, the best first filial (F.sub.1) hybrids have a substantial yield advantage over the best open-pollinated varieties or inbred lines. This yield advantage of a hybrid over its parents is termed "heterosis." The observed degree of heterosis varies among species; however, as a general rule, it is high among cross-pollinated species, such as maize and sunflower, and typically lower among self-pollinated species, such as soybean and wheat.
In this regard, from at least about the 1940's, hybrid varieties of maize (a/k/a corn) largely supplanted open-pollinated varieties because startling improvements in yield, along with other agronomic traits, were realized when hybrid varieties were used. Indeed, the manufacture and sale of hybrid seed are the basis of a significant agricultural industry.
To obtain F.sub.1 hybrid seed, it is necessary to cross two inbred parents. Although it is possible to do this via controlled pollinations (i.e., fertilizations) flower by flower, such an approach is labor-intensive and, thus, very expensive. Only high-value crops, such as ornamental flowers and the like, could absorb such production costs.
In maize, an intermediate course historically has been taken. Two maize parents have been grown in isolation from other potential maize parents, i.e., sources of pollen. One parent was detasseled (emasculated) and served as the "female" parent, whereas the other parent was allowed to produce pollen and fertilize the female parent by cross-pollination, thereby serving as a "male" parent. Which maize parent was chosen to be the female and which maize parent was chosen to be the male were frequently based on commercially significant reasons. For example, it was preferred to use maize with ample seed production as a female parent and maize with ample pollen production as a male parent. Hybrid seed was then harvested from the female parent.
The use of detasseling to emasculate a plant to provide a female parent for hybrid seed production continued until the discovery of cytoplasmic male sterility ("CMS"). CMS obviated the need to detassel because the products of genes involved in CMS caused ill-formed anthers that did not produce viable pollen. The causative factors of CMS have been shown to reside in the plant cell's cytoplasm (see Laughnan et al., Ann. Rev. Genet., 17, 27-48 (1983)). Such factors have been determined to be associated with mitochondria, which are maternally inherited. Accordingly, a cytoplasmic male-sterile line, which is incapable of cross- or self-pollination, can be used as a female parent. By crossing such a female parent with a pollen-producing line, fertile hybrid seed can be generated without detasseling the female plants.
An inbred line of maize can be converted into a CMS line by crossing it (as male) to a known cytoplasmic male-sterile line and then backcrossing it (as female) to the original inbred line. Given that the CMS-converted line is male-sterile, it must be maintained by crossing by the original inbred line (a "maintainer"). Hybrid seed is produced by growing the CMS-converted inbred line and a second inbred line in isolation, without detasseling.
Cytoplasmic male-sterile maize can be restored to fertility in a succeeding generation by a nuclear restorer gene. For example, if a CMS-converted line is grown in isolation with a second inbred line carrying a nuclear restorer gene, then the F.sub.1 will be male-fertile and potentially economically valuable.
There are three types of male-sterile cytoplasms in maize (Zea mays L. ): S (USDA), C (Charrua), and T (Texas). These three male-sterile cytoplasms can be distinguished by the ability of different nuclear (restorer) genes to restore fertility to the plants with these different cytoplasms (see Laughnan et al. (1983), supra), by mitochondrial DNA restriction endonuclease profiles (see Pring et al., Genetics, 89, 121-136 (1978)), and by .sup.35 S-methionine-labeled polypeptides translated in isolated mitochondria (see Forde et al., PNAS USA, 75, 3841-3845 (1978)).
In contrast to the male-sterile cytoplasms, the normal (N), male-fertile cytoplasm yields fertile plants in either the presence or absence of all known nuclear backgrounds, whereas the male-sterile C, S, and T cytoplasms only produce fertile plants in nuclear backgrounds carrying the appropriate restorer genes. These nuclearly encoded, fertility-restorer genes compensate for cytoplasmic dysfunction(s) that are phenotypically expressed during microsporogenesis and/or microgametogenesis. Plants carrying S or C cytoplasm are restored to fertility by a single dominant allele of the rf3 or rf4 locus, respectively. Preliminary evidence suggests that the rf3 locus is flanked by whp and bn117.14 on chromosome 2L (Kamps et al., Maize Genet. Coop. Newsl., 66, 45 (1992)). The rf4 locus maps to chromosome 8, approximately 2 cM from the RFLP ("restriction fragment length polymorphism") marker NP1114A (Sisco, Crop Sci., 31, 1263-1266 (1991)). In contrast to S and C cytoplasms, plants with T cytoplasm are restored to fertility by the dominant alleles of two loci, rf1 and rf2 (Laughnan et al. (1983), supra; and Levings et al., Plant Cell, 5, 1285-1290 (1983)), which are located on separate chromosomes. The rf1 locus is flanked by umc97 and umc92 on chromosome 3, and the rf2 locus is flanked by the umc153 and sus1 on chromosome 9 (Wise et al., Theor. Appl. Genet., 88, 785-795 (1994)).
T cytoplasm is restored to fertility at the sporophytic level; the genetic constitution of the diploid, sporophytic anther tissue, rather than that of the haploid, gametophytic pollen grain, determines pollen development. Therefore, a T-cytoplasmic plant, which is heterozygous for both restorer gene loci (Rf1/rf1, Rf2/rf2), will produce all normal pollen even though only one-fourth of the pollen grains carry both Rf1 and Rf2 (Laughnan et al. (1983), supra). In contrast, S-cytoplasm is restored to fertility at the gametophytic level, and, therefore, an S-cytoplasmic plant, which is heterozygous for rf3 (Rf3/rf3), will produce half normal pollen because only one-half of the pollen grains carry Rf3 (Laughnan et al. (1983), supra; and Schardl et al., Cell, 43, 361-368 (1985)).
CMS also occurs in other species of plants. Examples of other species of plants include petunia (Nivison et al., Plant Cell, 1, 1121-1130 (1989)), the common bean (Janska et al., Genetics, 135, 869-879 (1993)), Brassica napus (Singh et al., Plant Cell, 3, 1349-1362 (1991)), sunflower (Laver et al., The Plant Journal, 1, 185-193 (1991)), sorghum (Bailey-Serres et al., Theor. Appl. Genet., 73, 252-260 (1986)), and oats (Mann et al., Theor. Appl. Genet., 78, 293-297 (1989)). Like S-cytoplasmic maize, cytoplasmic male sterility in petunia, beans, and Brassica can be restored to fertility by single dominant nuclear genes.
Most of the research on CMS has focused on the characterization of novel open reading frames present in the mitochondrial genomes of male-sterile cytoplasms. Such research has revealed that, although each open reading frame is unique, all appear to have large hydrophobic domains (Dewey et al., PNAS USA, 84, 5374-5378 (1987)).
In T-cytoplasmic maize, CMS is associated with the unique mitochondrial gene T-urf13 (Wise et al., PNAS USA, 84, 2858-286 (1987a)). Toxin sensitivity traits are also associated with this gene (Huang et al., EMBO, 9, 339-247 (1990)). T-urf13 encodes a 13 kDa mitochondrial polypeptide (URF13) (Wise et al., Plant Mol. Biol., 9, 121-126 (1987b)), which is located in the mitochondrial membrane (Dewey et al. (1987), supra) and appears to span the mitochondrial membrane in oligomeric form (Korth et al., PNAS USA, 88, 10865-10869 (1991)). URF13 is not synthesized by deletion mutants (Dixon et al., Theor. Appl. Genet., 63, 75-80 (1982)), is truncated in the T4 frameshift mutant (Wise et al. (1987b), supra), and binds to fungal pathotoxins (Braun et al., Plant Cell, 2, 153-161 (1990)).
The abundance of URF13 is reduced by approximately 80% in plants carrying Rf1 and Rf2 (Dewey et al. (1987), supra). Also, there is an additional 1.6 kb T-urf13-specific transcript in such plants (Kennell et al., Mol. Gen. Genet., 210, 399-406 (1987)). The alteration of T-urf13 transcript accumulation and the concurrent reduction of URF13 appear to require the action of Rf1 only (Dewey et al. (1987), supra); however, other modifiers also appear to have an effect on T-urf13 transcript accumulation, depending on the nuclear background (Kennell et al. (1987), supra). Little is known about Rf2 except that, in addition to Rf1, it is essential for pollen restoration.
T cytoplasm was used predominantly in the late 1960's because of its reliability. The other male-sterile cytoplasms of maize, namely C and S, tended to "break down" in the field, i.e., self-pollination or incomplete fertility restoration occurred. Thus, approximately 85% of the hybrid maize seed in the U.S. was T-cytoplasm until the epidemic of southern corn leaf blight, which occurred in 1970 (Pring et al., Ann. Rev. Phytopathol., 27, 483-502 (1989)).
After the 1970 epidemic, it was determined that T-cytoplasmic maize is highly sensitive to the host-selective toxin (T toxin) produced by race T of the fungus Cochliobolus heterostrophus Drechsler (asexual stage Bipolaris mayadis Nisikado and Miyake), which is the causal organism of southern corn leaf blight (Comstock et al., Phytopathology, 63, 1357-1361 (1973)). T-cytoplasmic maize was also found to be highly sensitive to the host-selective toxin (Pm toxin) produced by another fungus, namely Phyllosticta maydis, Arny and Nelson, which causes yellow leaf blight (Yoder, Phytopathology, 63, 1361-1366 (1973)).
In view of the above, the major seed producers in the U.S. now use various combinations of male-sterile cytoplasms (including T). The use of various combinations of male-sterile cytoplasms enables a farmer to sow his fields with seeds of, for example, T, C, S, and N cytoplasms, wherein only those plants of N cytoplasm might entail detasseling.
A focus of research since the 1970's has been to develop alternative genetic approaches to emasculating plants for the purpose of hybrid seed production. This effort, in part, reflects a desire among farmers to maintain some level of genetic heterogeneity for any given crop. One approach (Marc Albertsen, Pioneer Hi-Bred International, Inc.) involves the use of nuclear male-sterile genes. This particular approach, which is predicated, at least in part, on earlier analogous work with Arabidopsis (see Aarts et al., Nature, 363, 715-717 (1993)), specifically uses a cloned nuclear male-sterile gene from maize, although there are a number of such genes in a given plant species, including maize (Albertson et al., Can. J. Genet. Cytol., 23, 195-208 (1981)). An inbred line of maize, for example, which is homozygous for a mutant allele of a nuclear male-sterile (ms) gene, is genetically engineered to carry a construct comprising an inducible promotor, which, upon induction, allows expression of a wild-type ms gene. The inbred line is maintained in isolation, where it is sprayed with the inducer and allowed to self- and sib-pollinate. Hybrid seed is produced by growing the inbred line with a second inbred line, which carries a wild-type allele of the ms gene, in isolation and in the absence of inducer. Accordingly, the F.sub.1 is heterozygous and, therefore, fertile. This approach is disadvantageous, however, in that it requires maintenance of the male-sterile line and the use of an inducer.
Another approach involves the use of a construct comprising an RNase gene operably linked to a tapetum-specific promotor (Leemans et al., Plant Genetic Systems; and Leemans et al., Nature, 347, 737-741 (1990)). The RNase is active only in the anthers, where it kills the tapetum, i.e., a structure that normally nourishes pollen. Introduction of this construct, via transformation or backcrossing, into an inbred line results in heterozygous dominant male sterility. When the heterozygous dominant male-sterile line is crossed by a N-cytoplasmic line, the progeny segregate for is and N (i.e., normal). Tight linkage of an herbicide resistance gene to the RNase gene enables elimination of fertile segregants. In practice, the male-sterile line is crossed by a normal progenitor line. The resulting segregating progeny are grown in isolation with a second inbred line. The rows that carry the first inbred line are sprayed with an herbicide. The male-fertile progeny die, leaving only the male-sterile inbred plants from which hybrid seed can be harvested. This approach is disadvantageous in that it requires the use of an herbicide to eliminate fertile plants, the presence of which reduce overall yield.
In view of the above, it is evident that there remains a need for an efficient and economical method of producing hybrid seed in high yield with a low risk of disease. Accordingly, it is an object of the present invention to provide new materials and methods that will enable one to produce hybrid seed from new and existing cytoplasmic male-sterile lines without the disadvantages attendant materials and methods currently available in the art. It is another object of the present invention to provide a method of producing a variant of a cytoplasmic male-sterile plant. It is yet another object of the present invention to provide a method of suppressing cytoplasmic male sterility in the progeny of a cytoplasmic male-sterile plant. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.